Electrophysiological-based assays are used in a variety of applications, which include the detection of toxicants, drug screening, illuminating the mechanism of toxicity, neuronal injury, epilepsy studies and biosensing. Most systems used for such assays produce information in a relatively low-throughput manner. For example, patch clamp systems, have an extensive history of use in identifying specific perturbations in electrophysiological function; however, they are also well known for their extremely low throughput (<10 cells/day). On the other hand, microelectrode array (MEA) systems, which have concurrent access to both single-cell and network-level activity, are higher-throughput and less technique-dependent; however, due to their high cost and limited sample capacity (typically <5 samples/experiment), they are still, functionally, low throughput. Currently, MEAs are expensive and are typically offered in units with only one culture well (such that only one tissue sample/cellular network at a time can be studied). The use of only one culture-well severely limits the throughput with which MEAs can be used to interface and investigate electrically active cellular networks.
In contrast, multiwell culture plates and plate readers are commonly used instruments in the pharmaceutical industry and are extensively used for high throughput in-vitro assays, such as screening compounds or toxicants. However, apart from enabling imaging, such transparent plates have no other function than to act as supporting structures for cell cultures and media, which eliminates the possibility of using multiwell plates for electrophysiological investigations. If electrodes could be integrated into to these transparent plates, high throughput applications like network electrophysiology can be carried out in a standard format. Integrating microelectrodes into a standard format would additionally enable compatibility with machinery in place for analysis of multiwell plates like microscopy and cell counting.
King et al. and Maher et al. disclose electrode arrays integrated with multiwell plates but the electrodes used are macro-sized (4 mm wide, 1 cm long and 0.2 mm thick in the case of Maher et al.) stainless steel plates. King et al. discloses an electroporation application to introduce molecules into lipid vesicles of cell membranes and Maher et al. report stimulation of cells in-vitro for studying transmembrane potentials recorded with optical measurement techniques. These disclosures by King et al. and Maher et al., introduce electrodes into multiwell formats, but the large size of the integrated electrodes eliminate the possibility for any cell based assays that address both single and network level cellular activity. Thus, micro-scale electrodes are required for such an investigation and the invention provided herein addresses the novel integration of microelectrodes into multiwell plates, such as a multiwell culture plate, with a transparent substrate in an ANSI/SBS (American National Standards Institute/Society for Biomolecular Sciences, “Standards for Microplates”, 2004) compliant format. However, the integration of micro-scale electrodes or MEAs into transparent, large area multiwell plates presents significant manufacturing challenges.
To-date, MEAs have been fabricated in both two- and three-dimensional conformations on a myriad of different substrates including flexible materials, such as poly dimethyl siloxane (PDMS), and rigid substrates, like silicon and glass. Regardless of the application or material, many of these MEAs share one significant drawback, expensive manufacturing costs. This expense is derived primarily from the packaging and assembly of the device, which is required to connect micron-sized electrodes for cellular interfacing to millimeter-sized sockets and pads for electrical processing. Such differences in scale introduce intermediate, often manual, processing steps that significantly reduce the manufacturability of MEAs. Additionally none of these known processes is truly standard (eg. Complimentary Metal Oxide Semiconductor or CMOS process for computer chips) resulting in high processing costs.